-immediately after the binding of RNA Pol II, TFIIE and TFIIH are recruited to the initiation complex

-TFIIH phosphorylates the carboxy terminal domain (CTD) of RNA Pol II

-also functions as a helicase and unwinds the double helix

-unwinding to DNA and the phosphorylation of RNA Pol II is sufficient for the polymerase to initiate transcription and escape the core promoter

-TFIIE appears to serve as a conduit between developmentally regulated transcription factors and TFIIH

10

Stabilization of Transcription

-initiation of transcription is rather messy with the polymerase frequently aborting the process during the synthesis of the +1 to +9 bases

-transcription is stabilized once the polymerase reaches the +10 base

-activity of TFIIE and TFIIH are critical for RNA Pol II clearance of the promote

11

Past Assumptions about Regulatory Step in Transcription

-assumed that major regulatory step in transcription was the recruitment of RNA Pol II and the general transcription factor machinery (TFIIA, TFIIB, TFIID and TFIIH) to the core promoter

-idea is that once RNA Pol II is recruited to the core promoter transcription will initiate immediately and full-length mRNA transcripts will be generated

-recent evidence has demonstrated that RNA polymerase can be found bound to sequences very close to the core promoters of genes that are not actively transcribed

12

RNA Pol II Within sim Gene and Polymerase Pausing (graphics)

-consider RNA Pol II within single minded (sim) gene -this gene is expressed only within the developing embryonic mesoderm

-RNA Pol II is not found near the promoter in this tissue (no peak) because transcription is initiated immediately after recruitment to the core promoter

-in contrast, even though sim is not expressed in either the embryonic dorsal ectoderm or the neurogenic ectoderm RNA Pol II is found at the core promoter in both tissues (note the presence of the peaks

-suggests that in the case of the sim gene, RNA Pol II is recruited to the promoter irrespective of whether it will be transcribed or not

-if cell requires sim to be activated then RNA Pol II will clear (or escape) the promoter and will quickly transcribe the entire gene. In contrast

-if a cell requires that sim remain inactive then RNA Pol II will remain bound near the promoter

-even in the cases where a gene is inactive, RNA Pol II will transcribe the first 30-50 bases and will then stall. This is called Polymerase Pausing and it appears to be a key regulatory step during the process of transcription.

13

Chromatin Immunoprecipitation

-first step in this experiment is to treat the cells or tissue with a cross-linking reagent such as formaldehyde which preserves all DNA-protein interactions

-chromatin is then isolated away from the cell debris and purified

-using sonication the chromatin is broken into smaller fragments that are approximately 500bp in length and contain just a few nucleosomes

-next step involves isolating the DNA fragments that are bound by a protein of interest (such as RNA Pol II).

-antibodies against such a protein are added to chromatin mixture

-entire mixture is then incubated for a short period to allow for the antibody to the chromatin.

-chromatin-antibody complex can be separated from the remaining DNA fragments by incubation with sepharose beads that are coated with Protein A or Protein G

-these two factors will interact with the antibody very tightly

-the DNA fragments that do not contain the DNA binding protein of interest (and by extension the antibody) will not bind to the sepharose beads and will be removed during the washing steps

-once the chromatin-antibody fragments are purified the complex can be treated with chemicals that will remove the antibody and the proteins (reversing the cross-linking process).

-naked DNA fragments can be isolated and cloned into plasmid vectors

-sequence of the fragment can be then determined -at end of this procedure you will know the sequence of DNA that is bound by an individual DNA binding protein

14

Pause Button (PB)

-new element with in eukaryotic core promoter

-in Drosophila several thousand genes have stalled or paused promoters (RNA Pol II paused between the +30 and +50 base)

-Michael Levine has looked for sequences within genes that signal RNA Pol II to pause.

-identified several sequences that were highly enriched within stalled promoters when compared to actively transcribed genes.

-ex: Inr element is found 5.68 times within every 1000bp of an active promoter

-in contrast, there are approximately 14.48 Inr elements within each kilobase of genes with stalled promoters

-in addition to identifying known elements that are enriched he discovered a new component of the stalled core promoter

-the sequence is [G/T]CG[G/A][A/T]CG and it is called the “pause button”

-density of this element within each kilobase of actively transcribed genes is 0.67.

-in stalled promoters the density of this element rises to approximately 4.79.

-enrichment suggests that the presence of the PB element is important for polymerase pausing

-element is unlikely to be the only required element as the GAGA, inverted GAGA and Inr elements are also enriched within stalled promoters

15

Combinatorial Code for Polymerase Pausing

-initiation and elongation of transcription is dependent upon a combination of core promoter elements

-is no single combination of sites that are found in all genes that are active or those that are paused

-it does appear that the combination of GAGA, Inr and PB sites is a good indicator that RNA Pol II will pause at this promoter

-of the 165 genes that have all three of these sites 81% of them have been shown to be paused

-RNA Pol II can stall at promoters that lack a PB site

16

Combination that Lacks PB Site

-combination of a GAGA Inr and DPE element has the next highest predictive value

-of the 228 promoters that have this combination 69% have stalled promoters

-implication of these results is that additional sequence elements are likely to important in predicting whether or not a RNA Pol II will stall at the promoter.

-PB site is likely to be important for stalling RNA Pol II but it is not absolutely necessary or required for this to happen

17

Positional Information within Core Promoter (graphics)

-using genomic and bioinformatic methods the location of the GAGA, PB and Inr elements were mapped in relation to the position of the transcriptional start site

-within constitutively transcribed genes the GAGA and PB elements are located at random positions and there is a slight bias for the Inr element to be found at the transcriptional start site

-on the other hand, within the promoters of genes that are stalled the GAGA site is most commonly found approximately 100 bases upstream of the transcriptional start site, the Inr site is found at the position of the transcriptional start site and the PB site is located between the +1 and the +30 site

-since mere presence of these sites is not sufficient for stalling RNA Pol II, this result suggests that the relative position of the GAGA, PB, and Inr sites is a crucial factor in whether RNA Pol II will pause during transcription